Hematopoietic stem cells (HSCs) are primitive cells that generate all the formed elements of the blood and immune system. These cells are functionally defined based on their capacity for self-renewal divisions, which leads to the continuous generation of new HSCs over the lifetime of an animal, and by their potential for pluripotent hematopoietic differentiation. There are three possible general outcomes for the resulting daughter cells when a hematopoietic stem cell divides: (i) differentiation, (ii) self-renewal, or (iii) apoptosis. Despite the extensive study of HSCs, due to its relevance to bone marrow transplantation, gene therapy, and basic hematopoiesis, the mechanisms controlling these three tightly regulated outcomes are poorly understood.
Purification strategies for HSCs have been developed for both mouse [Spangrude et al., Science 241:58-62 (1988):(published erratum appears in Science 244(4908):1030 (1989)); Uchida et al., J.Exp.Med. 175:175-184 (1992)] and humnans HSCs [Zanjani et al., J.Clin.Invest. 93:1051-1055 (1994), see comments; Larochelle et al., Nat.Med. 2:1329-1337(1996); Civin et al., Blood 88:4102-4109 (1996)]. Most of these strategies use antibodies directed against various cell surface antigens and multiparameter cell sorting to isolate phenotypically defined cell populations. This approach has allowed isolation of murine stem cell populations of sufficiently high purity to allow reconstitution of irradiated recipients with less than 10 cells [Morrison et al., Proc.Natl.Acati.Sci.USA 92:10302-10306 (1995); Osawa et al., Science 273:242-245 (1996)], while considerably greater numbers of sorted human cells have been required to reconstitute xenogeneic recipients [Larochelle et al., Nat.Med. 2:1329-1337 (1996); Zanjani et al., Exp.Hematol. 26:353-360 (1998), see comments].
Hematopoietic stem cells also represent attractive targets for genetic modification since their progeny make up the entire spectrum of the hematopoietic system. Gene therapy involving stem cells is thus an expanding field that potentially has important applications in the treatment of a wide range of diseases [Nienhuis et al., Cancer, 67:2700 (1991)]. However, due to the inherent quiescent nature of stem cells, retroviral gene transfer is limited since stable integration requires cell division. Improved transduction of this target cell population is thus one of the major goals of current gene therapy research. In the mouse, gene transfer and repopulation with genetically-modified bone marrow stem cells following transplantation has been reported [Lemischka et al., Cell, 45:917 (1986) and Dick et al., Cell, 42:71 (1985)]. Whereas the level of stem cell gene transfer and expression are relatively modest, it has been sufficient to investigate effects of gene expression on hematopoiesis [Persons et al., Blood 90:1777 (1997)]. In humans, only an extremely low number of transgenic stem cells persist on a long-term basis [Brenner et al., Lancet, 342:1134 (1993) and Brenner, et al., Lancet, 341:85 (1993) and Rill et al., Blood, 84:380 (1994)]. Therefore there is a need for increasing the proportion of such transduced stem cells through ex vivo expansion following transduction and/or through in vivo selection approaches.
Most current protocols for transduction of stem cells employ in vitro liquid suspension culture with hematopoietic growth factors. It is now well established that culturing murine bone marrow cells for 4 days in the presence of defined concentrations of interleukin-3, interleukin-6, and stem cell factor does not adversely effect overall stem cell survival and function. However, expansion beyond this point has not proven to be beneficial and results in depletion of the reconstitution potential of the bone marrow graft. Cytokine-stimulated stem cells cultured in expansion conditions typically either undergo differentiation or programmed cell death (apoptosis). More mature populations such as the CFU-S and CFU-C, however, are capable of significant expansion in culture. However, these cells are distinct from stem cells and only provide short to moderate-term repopulating ability in transplanted mice. In humans, the long-term culture-initiating cell (LTC-IC) can be expanded in vitro with appropriate combinations and amounts of growth factors. LTC-ICs have recently been shown to be more mature than the SCID mouse repopulating cell (SRC) [Dick et al., Cell 42:71 (1985)]. SRCs are depleted in cultures that are more than 4 days old, which is consistent with the SRC being a more primitive cell type. More recently, culturing hematopoietic stem cells derived from the AGM (a pre-liver intraembryonic site) has been reported [Dzierzak et al. WO 98/12304, hereby incorporated by reference in its entirety]. However, the prior art teaches at most a four-fold expansion of human hematopoietic cells [Bhatia et al., J. Exp. Med., 186:619-624 (1997)].
The human MDR1 gene and its murine homologs were originally identified based on the ability of their expressed products, collectively referred to as P-glycoproteins (P-gps), to extrude a wide variety of cytotoxic drugs from the cell interior [Gros et al., Cell, 47:371-380 (1986) and Chen et al., Cell, 47:381-389 (1986)]. It is now known that the MDR1 gene belongs to a superfamily of transport proteins that contain a conserved ATP-binding cassette (ABC) which is necessary for pump function [Allikmets et al., Hum. Mol. Genet. 5:1649-1655 (1996)]. Numerous studies have clearly shown that P-gp expression plays an important role in the resistance of human tumor cells to cancer chemotherapy [Pastan and Gottesman, Annu. Rev. Med., 42:277-286 (1991)]. Considering that P-gps are also expressed in a wide variety of normal tissues, more recent studies have examined the normal physiologic functions of MDR1-like genes. Murine gene disruption experiments have demonstrated that expression of various P-gps is necessary for biliary excretion [Smit et al., Cell, 75:451-462 (1993)], maintenance of the blood-brain barrier [Schinkel et al., Cell, 77:491-502 (1994)], and elimination of drugs [Schinkel et al., Proc.Natl.Acad.Sci. USA, 94:4028-4033 (1997)]. P-gps can also mediate more general cellular functions including the translocation of lipids across the cell membrane [van Helvoort et al., Cell, 87:507-517 (1996)] and modulation of specific apoptosis pathways [Johnstone et al., Blood, 93:1075-1085 (1999) and Smyth et al., Proc.Natl.Acad.Sci.USA, 95:7024-7029 (1998)].
P-gp is expressed in a variety of hematopoietic cell types [Drach et al., Blood, 80:2729-2734 (1992)], including human CD34+ stem cells [Chaudhary and Roninson, Cell, 66:85-94 (1991)] and murine c-kit+ stem cells [Sorrentino et al., Blood, 86:491-501 (1995)]. Several lines of evidence suggest that P-gp expression is functionally conserved in hematopoietic stem cells.
Another ATP transport protein that contains a conserved ATP-binding cassette is the gene product of the Bcrp1/Mxr/Abcp gene (referred to as Bcrp and BCRP hereafter). The BCRP cDNA was originally cloned from several different human tumor cell lines that were resistant to multiple drugs including doxorubicin, topotecan, and mitoxantrone [Doyle et al., Proc.Natl.Acd.Sci.USA 95:15665-15670 (1998):(published erratum appears in Proc Natl Acad Sci USA; 96(5):2569 (1999)); Maliepaard et al., Cancer Res. 59:4559-4563 (1999); Miyake et al., Cancer Res. 59:8-13 (1999)]. A highly related mouse homologue (Bcrp1) was cloned from fibroblasts selected for multidrug resistance [Allen et al., Cancer Res. 59:4237-4241 (1999)]. In contrast to the structure of the MDR1 gene, which consists of two duplicated halves, the predicted structure of BCRP is that of a “half transporter”, with a single ATP binding cassette and transmembrane region. The expression pattern of BCRP is highly restricted in normal human tissues, with the highest levels of mRNA detected in the placenta, and much lower levels detected in adult organs [Doyle et al., Proc.Natl.Acad.Sci.USA 95:15665-15670 (1998):(published erratum appears in Proc.Natl.Acad.SciUSA. 96(5):2569 (1999)); Allikmets et al., CancerRes. 58:5337-5339 (1998)].
Hematopoietic stem cells can be identified based on their ability to efflux fluorescent dyes that are substrates for P-gp, such as Rhodamine (Rho) 123 [Spangrude and Johnson, Proc.Natl.Acad.Sci.SA, 87:7433-7437 (1990); Fleming et al., J. Cell Biol., 122:897-902 (1993); Orlic et al., Blood, 82:762-770 (1993); and Zijlmans et al., Proc.Natl.Acad.Sci.USA, 92:8901-8905 (1995)] and Hoechst 33342 [McAlister et al., Blood, 75:1240-1246 (1990); Wolf et al., Exp. Hematol., 21:614-622 (1993); and Leemhuis et al., Exp. Hematol., 24:1215-1224 (1996)]. One particular approach for purifying stem cells is based on Hoechst dye-staining of bone marrow cells to identify a minor fraction of side population (SP) cells that are highly enriched for repopulating activity [Goodell et al., J. Exp. Med., 183:1797-1806 (1996)]. This SP phenotype identifies a primitive subset of stem cells present in multiple mammalian species [Goodell et al., Nat. Med., 3:1337-1345 (1997)], and based on verapamil inhibition studies, may be due to expression of P-gp or another ABC transporter [Goodell et al., J. Exp. Med., 183:1797-1806 (1996)].
Methodology for enriching pluripotent stem cells in culture could have a major impact on treatment of blood and immune-system disorders. For example, bone marrow transplantation is often the only option for persons having hematopoietic and immune-system dysfunctions caused by congenital disorders and or chemotherapy or radiation therapy. In addition, enriching pluripotent stem cells should greatly enhance the treatment of immunodeficency disorders. Furthermore, the effectiveness of the treatment of blood diseases by ex vivo gene therapy, e.g., treating sickle cell anemia or thalassemia, could also be substantially enhanced. Therefore, expansion of primitive stem cells in culture should be a major advance for all aspects of bone marrow transplantation as well as gene therapy applications. Unfortunately, despite the clear need for such methodology, heretofore, it has not been realized.
In addition, whereas a recent report demonstrates that sorting for expression of the vascular endothelial growth factor receptor can enrich human stem cells to near purity [Ziegler et al., Science 285:1553-1558 (1999)], there still remains a general need for better and more specific markers of human HSCs.
The citation of any reference herein should not be deemed as an admission that such reference is available as prior art to the instant invention.